Hiroshi Yorifuji

Expression of a system L neutral amino acid transporter at the blood-brain barrier

Amino acid transport system L has been proposed to be one of the major nutrient transport systems at the blood–brain barrier. Using immunohistochemical analyses, a system L transporter LAT1 was shown to be expressed in the brain capillary endothelial cells in rats. Because LAT1 was co-expressed with 4F2 heavy chain which brings LAT1 to the plasma membrane, LAT1 is proposed to be functional in the plasma membrane of brain capillary endothelial cells. Both LAT1 and 4F2hc immunoreactivities were detected in a double line appearance surrounding endothelial cell nuclei, suggesting both proteins are present in the luminal and abluminal membranes. LAT1 is, thus, a blood–brain barrier system L transporter responsible for the permeation of aromatic or branched-chain amino acids and amino acid-related drugs such as L-DOPA.

Ethidium Bromide-induced Inhibition of Mitochondrial Gene Transcription Suppresses Glucose-stimulated Insulin Release in the Mouse Pancreatic β-Cell Line βHC9

Recently, a mitochondrial mutation was found to be associated with maternally inherited diabetes mellitus (Kadowaki, T., Kadowaki, H., Mori, Y., Tobe, K., Sakuta, R., Suzuki, Y., Tanabe, Y, Sakura, H., Awata, T., Goto, Y., Hayakawa, T., Matsuoka, K., Kawamori, R., Kamada, T., Horai, S., Nonaka, I., Hagura, R., Akanuma, Y., and Yazaki, Y. (1994) N. Engl. J. Med. 330, 962–968). In order to elucidate its etiology, we have investigated the involvement of mitochondrial function in insulin secretion. Culture of the pancreatic β-cell line, βHC9, with low dose ethidium bromide (EB) (0.4 μg/ml) for 2–6 days resulted in a substantial decrease in the transcription level of mitochondrial DNA (to 10–20% of the control cells) without changing its copy number, whereas the transcription of nuclear genes was grossly unaffected. Electron microscopic analysis revealed that treatment by EB caused morphological changes only in mitochondria and not in other organelles such as nuclei, endoplasmic reticula, Golgi bodies, or secretory granules. When the cells were treated with EB for 6 days, glucose (20 mm) could no longer stimulate insulin secretion, while glibenclamide (1 μm) still did. When EB was removed after 3- or 6-day treatment, mitochondrial gene transcription recovered within 2 days, and the profiles of insulin secretion returned to normal within 7 days. Studies with fura-2 indicated that in EB-treated cells, glucose (20 mm) failed to increase intracellular Ca2+, while the effect of glibenclamide (1 μm) was maintained. Our system provides a unique way to investigate the relationship between mitochondrial function and insulin secretion.

Morphological evidence of the inhibitory effect of taxol on the fast axonal transport

The short term effects of taxol, a stabilizing drug of microtubules, on the peripheral nerves in the rat was investigated using a new chamber system which can be applied to incubate a sciatic nerve with various solutions in vivo. A functional analysis of retrograde axonal transport using rhodamine-labeled wheat germ agglutinin (WGA-rhodamine) showed the inhibitory effect of the drug. An electron microscopic study also revealed that a variety of vesicles were observed to accumulate on both the proximal and the distal sides of the chamber, however, no significant increase in the number of microtubules in the axons, based on the pharmacological effect of the drug, was observed even though one had been expected. These findings support the inhibitory effect of taxol on the fast axonal transport of the neurons. Furthermore, the accumulated vesicles were morphologically different from those accumulated by ligation. These results suggest that a special component of the fast axonal transport was thus selectively blocked by the drug.

Expression of dystroglycan and laminin-2 in peripheral nerve under axonal degeneration and regeneration

Abstract In Schwann cells, the transmembrane glycoprotein β-dystroglycan composes the dystroglycan complex, together with the extracellular glycoprotein α-dystroglycan which binds laminin-2, a major component of the Schwann cell basal lamina. To provide clues to the biological functions of the interaction of the dystroglycan complex with laminin-2 in peripheral nerve, the expression of β-dystroglycan and laminin-α2 chain was studied in rat sciatic nerves undergoing axonal degeneration and regeneration as well as in normal condition. In normal sciatic nerve, immunoreactivity for the cytoplasmic domain of β-dystroglycan was consistently and selectively localized in the Schwann cell cytoplasm underlying the outer (abaxonal) membrane apposing the basal lamina. While β-dystroglycan expression was gradually down-regulated in Schwann cells losing contact with axons during axonal degeneration, it was progressively up-regulated as the regenerating process of ensheathment and myelination proceeded during regeneration. Interestingly, β-dystroglycan expression, when detectable, was always restricted to the Schwann cell cytoplasm beneath the outer membrane apposing the basal lamina during both axonal degeneration and regeneration. Furthermore, laminin-α2 immunoreactivity roughly paralleled that of β-dystroglycan during both axonal degeneration and regeneration, indicating that the expression of β-dystroglycan and laminin-α2 is induced and maintained by the Schwann cell contact with axons. Our results indicate that the dystroglycan complex is involved in the adhesion of the Schwann cell outer membrane with the basal lamina and suggest that the dystroglycan complex may play a role in the process of Schwann cell ensheathment and myelination through the interaction with laminin-2.

Neurofilaments of Klotho, the mutant mouse prematurely displaying symptoms resembling human aging

We reported previously that neurofilaments (NFs) of aged rats were highly packed in the axon and contained a smaller amount of NF‐M as compared with those of young rats (Uchida et al. [ 1999 ] J. Neurosci. Res. 58:337–348). We studied NFs of the mutant mouse, named Klotho, which displays prematurely symptoms resembling human aging. The transport of axonal cytoskeletal proteins, including NFs, tubulin and actin, was decreased at the leading portion of the peak of transported proteins in Klotho during the process of premature aging. The nearest neighbor inter‐NF distance in Klotho axons (35–39 nm) was shorter than that of the wild‐type mouse (48–49 nm), indicating the packing of NFs in Klotho. The ratio of NF‐M to NF‐L was slightly decreased in cytoskeletons from the spinal cords of Klotho. These changes are similar, though not identical, to those observed in aged rats, and are the first evidence of age‐related changes in the neurons of Klotho. J. Neurosci. Res. 64:364–370, 2001.

Expression of Dystroglycan and the Laminin-α2 Chain in the Rat Peripheral Nerve during Development

In Schwann cells, the transmembrane glycoprotein beta-dystroglycan comprises the dystroglycan complex, together with the extracellular glycoprotein alpha-dystroglycan, which binds laminin-2 (alpha 2/beta 1/gamma 1), a major component of the Schwann cell basal lamina. To provide clues to the biological functions of the interaction of the dystroglycan complex with laminin-2 in peripheral nerves, we investigated the expression of beta-dystroglycan and the laminin-alpha 2 chain in rat sciatic nerve during development by immunoblot, immunofluorescence, and immunoelectron microscopic studies. The expression of beta-dystroglycan and the laminin-alpha 2 chain in the rat sciatic nerve was low and not confined to the Schwann cell outer membrane from embryonic day 18 to birth, when there was only an immature basal lamina assembly and no compact myelin formation by Schwann cells. However, the expression of these proteins increased markedly and became clearly localized to the Schwann cell outer membrane between birth and postnatal day 7, when both basal lamina assembly and compact myelin formation by Schwann cells progressed rapidly. From postnatal day 7 to adult, there was no remarkable change in the expression of these proteins. Our results support the hypothesis that the dystroglycan complex functions as an adhesion apparatus, binding the Schwann cell outer membrane with the basal lamina, and suggest that the dystroglycan complex plays a role in Schwann cell myelination through its interaction with laminin-2.

Centrally nucleated fibers (CNFs) compensate the fragility of myofibers in mdx mouse

Centrally nucleated fibers (CNFs) are the myofibers which have nuclei in the center of cytoplasm, and are generally recognized as regenerated myofibers. They are commonly observed in the histopathology of the patients with several types of muscular dystrophies and their animal models. In the mdx mouse, an animal model of Duchenne muscular dystrophy, CNFs are more resistant than non-CNFs to mechanical stresses, as evidenced by the Evans blue infiltration. In relation to the population among muscles, CNFs are supposed to compensate the fragility of muscular tissue in muscular dystrophies and their animal models.

Comparative effects of linoleic acid and linoleic acid hydroperoxide on growth and morphology of bovine retinal pigment epithelial cells in vitro

PURPOSE Outer segments of the photoreceptor rods that are phagocytized by the retinal pigment epithelial (RPE) cells contain a high proportion of polyunsaturated fatty acids (PUFA). PUFA are susceptible to lipid peroxidation. We hypothesized that the resulting peroxides could injure RPE cells leading to retinal degeneration. Accordingly, we compared the effects of linoleic acid (LA) and its hydroperoxide (LHP) on the growth and morphology of RPE cells using laser scanning microscopy and transmission microscopy. METHODS We counted the number of RPE cells after incubation for 24 and 48 hrs with concentrations of LA or LHP of 0.035, 0.175, and 0.35 mM. To observe the actin filaments, cultured RPE cells were stained with rhodamine phalloidin. The cells were prefixed with 2% glutaraldehyde and postfixed in 1% osmium tetroxide. Specimens were embedded in Epon 812 after dehydration, and the ultrathin sections were doubly stained with 2% uranyl acetate and 2% lead acetate for examination by transmission electron microscopy. RESULTS Exposure to LA or LHP produced dose-dependent damage to RPE cells with a significantly greater effects of LHP than LA. After incubation for 24 hrs with 0.35 mM LA, the number of vacuoles in RPE cells exceeded that observed in control RPE cells by 365 nm laser microscopy. Exposure to 0.35 mM LHP for 24 hrs produced a pycnotic nucleus, with diffuse and granular autofluorescences observed in and around it. Exposure of RPE cells to 0.35 mM LA for 24 hrs showed that the LA incorporated into the lysosomes was digested and released extracellularly from lysosomes via exocytotic vesicles. However, such exposure to LHP damaged the RPE cells, including the membranes in the pinocytotic vesicles. The packed membranes resembled myelin. CONCLUSIONS While the LA incorporated into the lysosomes was released extracellularly, LHP persisted in the RPE cells, being observed as autofluorescent lipofuscin-like materials. LHP was cytotoxic, and caused damage to the membranes of pinocytotic vesicles and lysosomes.

Neurofilaments of Aged Rats: The Strengthened Interneurofilament Interaction and the Reduced Amount of NF-M

Amyotrophic lateral sclerosis is an age‐related neurological disease, characterized by neurofilament (NF) accumulation in primary axons followed by degeneration of motor neurons. To elucidate age‐related factors that might lead to pathological NF accumulation, NFs were compared between young and aged rats. Electron microscopic examination of sciatic nerve axons revealed that NFs were more than twice as densely packed in aged rat axons (542 ± 180 NFs/mm2) as in young adult rat axons (211 ± 73 NFs/mm2). The NFs isolated from aged rats also appeared to be more aggregated than those from young rats. Phosphorylation at the head or tail domains was studied as a possible candidate affecting NF organization. Western blotting with phosphorylation‐dependent antibodies showed higher phosphorylation of NF‐H in the tail domains of aged rat spinal cord NFs, but dephosphorylation did not diminish the differences in aggregation between aged and young rat NFs. On the other hand, when NFs were phosphorylated by A‐kinase on their head domains, the extent of phosphorylation in NF‐M of aged rat NFs was only one‐third of young rat NFs. We found that aged rat NFs contained only 60% of the NF‐M of young rat NFs in molar ratio compared to NF‐L. These results raise a possibility that the decreased amount of NF‐M induces the aggregates of isolated NFs and the higher packing density of NF in aged rat axons. J. Neurosci. Res. 58:337–348, 1999.

Expression of dystroglycan complex in satellite cells of dorsal root ganglia

Abstract. In Schwann cells, the transmembrane glycoprotein β-dystroglycan composes the dystroglycan complex together with the extracellular glycoprotein α-dystroglycan, which binds laminin-2 (α2/β1/γ1), a major component of the Schwann cell basal lamina. In the Schwann cell cytoplasm, β-dystroglycan is anchored to a dystrophin isoform, Dp116. In this study, we investigated the expression of β-dystroglycan, Dp116 and the laminin-α2 chain in satellite cells of rat dorsal root ganglia (DRGs). Immunohistochemical study showed that immunoreactivities for β-dystroglycan and Dp116 were both localized to the outer rim of neuron-satellite cell and axon-Schwann cell units, indicating that both satellite and Schwann cells expressed these proteins in DRGs. Immunoreactivity for the laminin-α2 chain was detected in a similar location, indicating that the basal lamina surrounding satellite and Schwann cells in DRGs contained laminin-2. Ultrastructurally, immunoreactivity for the cytoplasmic domain of β-dystroglycan as well as that for Dp116 was most intense in the cytoplasm just underlying the outer membrane of satellite cells. The immunoreactivity for laminin was associated with the outer surface of those cells, suggesting that it was localized in the surrounding basal lamina. These results indicate that the dystroglycan complex is expressed in the satellite cell outer membrane and involved in the adhesion with the basal lamina through the interaction with laminin-2.